Automated polypeptide synthesis apparatus

ABSTRACT

An apparatus is provided for automatically constructing a polypeptide of high purity, up to 50 amino acids in length, using only single couplings. The apparatus includes an activation system for receiving protected amino acids, one kind at a time, having a common vessel (an activator vessel) in which to activate each of the amino acids. Also included is a reaction vessel for containing a resin used in solid-phase peptide synthesis for attaching a peptide chain thereto. A transfer system is also provided, which operates under control of a computer, to transfer the activated species from the activation system to the reaction vessel and to transfer amino acids, reagents, gases, and solvents from one part of the apparatus to another. The activator system also includes a temperature controlled concentrator vessel in which an activator solvent is replaced by a coupling solvent to enhance the coupling of the activated species to the peptide chain in the reaction vessel. Also included in the synthesizer system is a vortexer for affecting total washing of materials in the reaction vessel and the reaction vessel itself, an automated peptide resin sampling system, and an autodelivery system for providing individual containers of amino acid to the synthesizer in the order desired in the peptide sequence. A liquid sensor system is also included to monitor transitions between gases and liquids in specific tubes in the synthesizer in order to provide input signals to the computer system for control purposes. The computer system software which controls the operation of the synthesizer is organized according to a series of menus which allows the user of the system to select individual cycles of operation for each vessel in the synthesizer. In addition, an algorithm has been developed which provides for optimum efficiency in tbe production of a peptide for any given selection of cycles.

CROSS REFERENCE TO RELATED APPLICATION

This is a division of application Ser. No. 07/053,324, filed May 22,1987, which is a division of Ser. No. 06/592,638, filed Mar. 23, 1984,issued U.S. Pat. No. 4,668,476, issued May 26, 1987.

FIELD OF INVENTION

This invention relates to apparatus for the automated synthesis ofpolypeptides, and particularly to apparatus for automaticallypre-forming activated species of (alpha-amino protected) amino acidsimmediately prior. to introduction into solid phase synthesis reactions.

BACKGROUND OF THE INVENTION

Since its inception in 1962, R. B. Merrifield's concept of solid phasepeptide synthesis has seen many improvements and has now become anestablished technique in the art. Literally hundreds of investigationshave been published describing the chemical details of the method (Seefor example, Merrifield, R. B.: Science 150, 178 (1965); Merrifield, R.B.: Sci. Amer. 218, 56 (1968); Stewart, J. M., Young, J. D.: In: SolidPhase Peptide Synthesis. San Francisco, Calif.: Freeman 1969; andErickson, B. W., Merrifield, R. B.: In: The Proteins (eds. Neurath, R.L. Hill), III. Ed., Vol. 2, pp 255-527. New York: Academic Press 1976).)

Typically, solid phase peptide synthesis begins with the covalentattachment of the carboxyl end of an (alpha-amino protected) first aminoacid in the peptide sequence through an organic linked to an insolubleresin bead (typically 25-300 microns in diameter), illustrated by:##STR1## A general cycle of synthesis then consists of deprotection ofthe resin bound alpha-amino group, washing (and neutralization ifnecessary), followed by reaction with with some carboxyl activated formof the next (alpha-amino protected) amino acid to yield: ##STR2##Repetition of the cycle to the nth amino acid then yields: ##STR3## Atthe end of the synthesis, the link of the peptide to its polymer supportis cleaved, and the dissolved peptide is separated from the insolubleresin and purified.

Although this process is simple in principle, in practice it can bequite difficult to obtain peptides over about 30 amino acids long whichhave any substantial purity. The reason for this is that the averagestep yield has a profound effect on the purity of the product peptide,as illustrated by the values in the following table for synthesis of a30 amino acid peptide.

                  TABLE                                                           ______________________________________                                        30 AMINO ACID PEPTIDE                                                         Average Step Yield (%)                                                                         Product Purity (%)                                           ______________________________________                                        95.0             21                                                           99.5             86                                                           99.7             91                                                           ______________________________________                                    

The results are even more problematic for longer peptides, eg. for atarget peptide with 101 residues, a step yield of 99.0% provides aproduct of only 36% purity. In all cases, the by-products of peptidesynthesis consist of a complex mixture of molecules which are chemicallysimilar to the target peptide. Chromatographic purification can beextraordinarily difficult and time consuming as the relative amount ofby-product molecules begins to exceed about 25%.

The efficiency of step yield is dependent on many factors such as thenature and quality of the protected amino acids, solvent purity,chemical integrity of the resin, the chemical nature of the organiclinker, the form of the activated carboxyl of the amino acid, efficiencyof the wash steps, the synthesis protocol, and in some instances theidentity of an amino acid in conjunction with a particular sequencesegment to which it is being added.

Each of the above factors, when not optimally controlled, willcontribute some significant increment to yield reduction in everycoupling step. At the present time, the complexity of these factors issuch that average step yields in solid phase peptide synthesis aretypically in the range of 93-97% for both manual and automatedexecutions. For practical applications on a commercially reasonablescale, such as for the development of pharmaceuticals, enzyme substratesand inhibitors, hormones, vaccines, and diagnostic reagents, such lowstep yields significantly increase costs of production and in many casesmake such direct solid phase synthesis of peptides impractical.

Prior art peptide synthesizers operate essentially as "washing machines"which automate the monotonous fluid manipulations of deprotection,addition of coupling agent, and washing. In no case do existingcommercial peptide synthesizers form an activated amino acid speciesoutside or independent of the reaction vessel. Typically, protectedamino acid and DCC are added to the reaction vessel containing the resinbound, incipient peptide chain so that activation of the amino acidoccurs in the presence of the deprotected alpha-amino group. Thisapproach both limits the possibility (or feasibility) of optimizingactivation conditions for individual amino acids and requires that anymodification of activation conditions be done in the presence of thedeprotected alpha-amino group and the growing, resin-bound peptidechain. This fact makes it difficult, if not impossible, to optimizeactivation parameters by analyzing rates of formation and relativethermal and solvent stabilities of the individual, activated amino acidspecies. Additionally, the ability to use various thermal inputs duringthe activation process can only be done in the presence of the peptidechain.

SUMMARY OF THE INVENTION

In accordance with the preferred embodiment of the invention, anapparatus is provided for automatically constructing a polypeptide ofhigh purity, up to 50 amino acids in length, using only singlecouplings. The apparatus includes an activation system for receivingprotected amino acids, one kind at a time, having a common vessel (anactivator vessel) in which to activate each of the amino acids in theorder received to form a sequence of aliquots of activated species ofeach of the amino acids, each aliquot containing one kind of amino acidand the sequence of aliquots of each kind of amino acid being in theorder desired in the peptide. Also included is a reaction vessel forcontaining a resin used in solid-phase peptide synthesis for attaching apeptide chain thereto. A transfer system is also provided, whichoperates under control of a computer, to transfer the activated speciesfrom the activation system to the reaction vessel and to transfer aminoacids, reagents, gases, and solvents from one part of the apparatus toanother. The activator system also includes a temperature controlledconcentrator vessel in which an activator solvent, which is used in theactivator vessel when creating the activated species of the amino acid,is replaced by a coupling solvent to enhance the coupling of theactivated species to the peptide chain in the reaction vessel. Thisreplacement is accomplished a short period of time (typically less thanthirty minutes) before the activated amino acid is introduced into thereaction vessel, by adding the coupling solvent to the concentratorvessel together with the activated species and the activator solvent,and sparging gas through the resulting solution to selectively evaporatethe activator solvent, activator solvent being chosen with a boilingpoint lower than the boiling point of the coupling solvent. Theconcentrator is heated as necessary to replace heat lost by evaporation.

Also included in the synthesizer system is a vortexer for affectingtotal washing of materials in the reaction vessel and the reactionvessel itself, an automated peptide resin sampling system, and anautodelivery system for providing individual containers of amino acid tothe synthesizer in the order desired in the peptide sequence. Also, aspecialized container for use in the autodelivery system is providedwhich has a vee-shaped bottom in order to permit extraction of as muchamino acid as is practicable which permits precise control overstoichiometry. A liquid sensor system is also included to monitortransitions between gases and liquids in specific tubes in thesynthesizer in order to provide input signals to the computer system forcontrol purposes.

The computer system software which controls the operation of thesynthesizer is organized according to a series of menus which allows theuser of the system to select individual cycles of operation for eachvessel in the synthesizer. In addition each cycle can be user-definedinto a series of functions, each of which corresponds to a standard setof instructions for individual valves and other switched systemsassociated with the synthesizer. Also, using the menu system, the usercan define alternative individual functions as well.

In addition to the menu driven control system, an algorithm has beendeveloped which is related to the organization of the computer softwareinto individual cycles for each vessel. Operating the synthesizeraccording to the algorithm provides for optimum efficiency in theproduction of a peptide for any given selection of cycles.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a and 1b illustrate the fluid delivery system of the apparatusaccording to the invention.

FIG. 1A illustraes how FIGS. 1a and 1b are to be arranged to illustratethe fluid delivery system.

FIG. 1B illustrates gas sources that are connected through manifolds andregulators to indicated inputs of the elements of FIGS. 1a and 1b.

FIG. 2 illustrates the computer system used for controlling theapparatus.

FIG. 3 is a top view of an autodelivery system for providing individualcontainers to the synthesis apparatus.

FIG. 4 is a cross-sectional view of a reaction vessel according to theinvention showing the results of vortexing on fluids contained therein.

FIGS. 5a, 5b, and 5c are three views of a container used in theautodelivery system.

FIG. 5d is a bottom view of the container illustrated in FIGS. 5a-5c.

FIG. 6 is a table showing the dimensions of the container used in theautodelivery system.

FIGS. 7a and 7b show two views of a liquid sensor used in the apparatus.

FIGS. 8-16 illustrate various menus used in the computer system todefine operations according to which to run the synthesizer apparatus.

FIGS. 17a and 17b illustrate the menu flow scheme.

FIG. 17 illustrates how FIGS. 17a and 17b are to be arranged toillustrate the menu flow scheme.

FIG. 18 is a diagram to provide definitions for use in the calculationalscheme for optimizing production of peptide.

FIG. 19 provides an example of a calculation for optimizing theproduction of a peptide which requires three cycles of coupling.

FIGS. 20a and 20b compare prior yields with the yields produced by theapparatus disclosed herein.

DETAILED DESCRIPTION OF THE INVENTION

Before the details of the apparatus of the invention are described, itis useful first to understand the chemical approach of the solid phasepeptide synthesis which the apparatus is designed to optimize.

For the present invention, the preferred mode of the synthesis takesplace primarily in three phases. The first, or activation phase involvesthe production of the (alpha-amino protected) amino acid symmetricanhydride as the acylating species: ##STR4## Symmetric anhydrides areextremely effective, activated carboxyl forms of amino acids, sincetheir couplings are substantially free of racemization in the absence ofbase and since quantitative single couplings are usually assured formost amino acid additions, with the exception of asparagine, glutamine,and arginine--each of which is more efficiently coupled by analternative activation method which will be discussed later. Thegenerally quantitative nature of couplings with symmetric anhydridesmakes them most useful where automated synthesis precludes convenientstep by step quantitative monitoring. The apparatus herein describedautomatically synthesizes symmetric anhydrides immediately beforeincorporation into the peptide chain. Because of the marginal stabilityof symmetric anhydrides and their difficulty of isolation in pure form,their use in the past has been limited to manual preformation followedby introduction into an automated synthesis machine.

The procedure for synthesizing pre-formed symmetric anhydrides (PSA's)consists of reacting 0.5 equivalents of dicyclohexylcarbodiimide (DCC)with 1.0 equivalent of protected amino acid in dichloromethane (DCM)according to the equation: ##STR5## dichloromethane being an optimalsolvent for the synthesis of PSA's, particularly where the alpha-aminoprotecting group P is the t-butyloxycarbonyl group (t-BOC). The DCU,formed in the reaction, however, is very insoluble in dichloromethaneand precipitates during the PSA reaction. After completion of thereaction, the PSA/DCM solution is filtered away from the DCUprecipitate, and the second, or concentration phase is begun. In theconcentration phase the DCM is removed and replaced by polar aproticsolvent, preferably N,N-dimethylformamide (DMF), to enhance couplingefficiency during later solid phase reactions. The third, or reactionphase then follows the general schema described in the Background of theInvention whereby to attach an additional amino acid to the sequence thecarboxyl end of the PSA is reacted with an alpha-amino deprotectedresin-bound peptide chain.

Apparatus

In accordance with the preferred embodiment of the invention, anapparatus for achieving the s thesis described above is illustrated inFIGS. 1a, 1b, 1B, 2 and 3, which show respectively a fluid deliverysystem, for routing the various amino acids, reagents, solvents andgases throughout the apparatus; a computer system for effectingautomatic control over the numerous switches which control the valves,sensors, temperature of certain vessels, and motors in the apparatus;and an autodelivery system for transporting protected amino acids to theapparatus in the order desired in the peptide sequence.

The fluid delivery system includes three primary vessels: an activatorvessel 11, where the PSA is formed; a concentrator vessel 13, where thePSA/DCM solution from the activator vessel is transferred so that DCMmay be replaced by DMP to enhance coupling; and a reaction vessel 15which contains the growing resin-bound peptide chain.

Activator vessel 11 is typically cylindrical, about 40 ml in volume, andis preferably constructed of glass in order for the operator of thedevice to visually inspect the progress of reactions or cleaning cycles.At the bottom of the activator vessel is a glass frit 17 of coarseporesize which is used to filter the DCU precipitate from the PSA/DCMsolution when transferring the solution to the concentrator vessel 13.Activator vessel 11 also contains an overhead nozzle 19 which facesupward in order to achieve a total washdown of the headspace and wallsafter each amino acid is transferred out of the vessel. Activator 11 iscoupled to the autodelivery system and to various gases and reagents asshown via a valve block 23, which is an assembly of zero dead volumevalves such as that described in U.S. Pat. No. 4,008,736, issued Feb.22, 1977, entitled VALVE ARRANGEMENT FOR DISTRIBUTING FLUIDS, byWittman-Liebold, et al., as are all other valve blocks in the system.Valve block 23 is operated under the control of the computer system, asare all other valve blocks and gas valves in the apparatus. Activator 11is coupled via nozzle 19 to another valve block 25 which controls theflow of methanol and DCM into the vessel for dissolving DCU precipitatefor cleaning and which controls the pressure inside the vessel to effecttransfers of materials into and out of the vessel from block valve 23.Transfers from the bottom of the vessel take place through a translucenttube 29, typically constructed of Teflon™, the transfers being monitoredby the computer system by means of a liquid sensor 27 which detectstransitions in tube 29 between gases and liquids. Typically tube 29, andother tubes in the system to which similar liquid sensors are attachedhave a roughly calibrated flow resistance and operate at a fixed knownpressure during transfers, so that the length of time required for atransfer corresponds directly to the volume of material which istransfered. For DCC and HOBT, which require specific volumes, acalibrated delivery loop is used to achieve a higher accuracy. Hence,the computer system can accurately monitor all flow into and out of theactivator vessel. Although details of the liquid sensor will bedescribed later, it is important to emphasize that the use of liquidsensors is not required for operation of the apparatus. They are usefulhowever in achieving better system control.

The next major section of the fluid delivery system is the concentratorvessel 13. Its construction is substantially the same as that ofactivator vessel 11, and includes an overhead nozzle 31, and a glassfilter frit 35 at the bottom. In addition, however, the concentratorvessel also has a band heater 37 attached thereto, which is used tocontrol the temperatures inside the vessel through the use of athermistor as the DCM in the PSA/DCM solution is replaced by DMF.Attached to concentrator vessel 13 is a valve block 33, and atranslucent transfer tube 39 monitored by a liquid sensor 40. Transfersthrough tube 39 are controlled by the computer system by means of valveblock 41.

The next major section of the fluid delivery system is the reactionvessel 15, which in the preferred embodiment is a right circularcylinder oriented vertically and is constructed of a machinedfluorocarbon polymer, such as Teflon™, or KEL-F™. The vessel is valvedat the top by a valve block 43 and at the bottom by a valve block 45,each valve being isolated by a filter such as membrane 47 and membrane49 which are typically constructed of a material such as ZITEX™,produced by Chemplast Inc. of Wayne, N.J., although glass frits couldalso be used. The reaction vessel is designed to be opened conveniently,both for initial charging with the loaded resin and for periodic removalof sample aliquots. This is accomplished by threading the top and bottomof the reaction vessel cylinder to accommodate threaded caps. Thethreaded caps are also used to hold the membranes in place, and each capis configured to accept a tube in order to transfer fluids into and outof the reaction vessel. A typical volume for the reaction vessel canvary widely, depending on the length of the peptide chain to besynthesized and the weight and amino acid loading of the synthesisresin. For example, for chains up to 50 amino acid units in length,starting with 0.5 grams of resin (0.5 m mole of amino acid), a preferredsize is about 40 ml. Those skilled in the art of solid phase synthesiswill realize that the resin may swell from three to five fold due tosolvent imbibement during synthesis. Mass increase as a result of growthof the peptide chain can cause an increase in the occupied volume of thereaction vessel from 10% initially to as much as 80% at the end of thesynthesis, depending on the length of the peptide.

In order to promote efficient coupling and to avoid agglomeration of theresin beads it is important to agitate the reaction vessel at variousstages in the reaction cycle. Also, it is especially important that theentire inner surface of reaction vessel 15 be completely rinsed duringeach wash cycle between the additions of PSA's from the concentratorvessel. To achieve this agitation, the bottom of the reaction vessel ismoved in a circle having a radius of about 0.093 in., about its centeraxis at about 1500 rpm, by a motor 48 (connected by a pully to aneccentric on the bottom of the reaction vessel) under control of thecomputer system, while the center of the top of the reaction vessel isheld substantially fixed, with the vessel itself being prevented fromrotating. The result is a conically rotational motion of the fluid resinmixture in the reaction vessel about the vertical axis which has theappearance of a vortex.

This "vortex" agitation mode enables use of very small volume incrementsof wash solvent for all washing operations, thus greatly improving theefficiency of removal of spent reagents and reagent by-products from thesynthesis resin, since it is much more efficient to extract thesematerials by successive partitioning, than by a single extraction. Theability to use small volume increments is a result of the angularmomentum of the fluid resin mixture imparted by the conical motion. This"vortexing" action creates a distribution of fluid in the vessel asdepicted in FIG. 4, wherein the fluid in the reaction vessel can be madeto contact all interior surfaces of the reaction vessel, for very smallvolume increments of the solvent by proper choice of the speed ofrotation. The result is more efficient washing of the resin by smallervolumes of expensive solvents.

Additionally, this mode of agitation prevents resin agglomeration andallows total fluid-resin interaction without the use of impeller typemechanical agitation. With mechanical agitation, the shear and resinabrasion caused by the impeller can fracture the resin beads intosmaller and smaller particles which can eventually clog the filters,such as membranes 47 and 49, thus forcing interruption of the synthesisprocess. Such an interruption can have dire effects on synthesis, e.g.restriction of flow (out of the Reaction vessel) could occur during anacid deprotection step, thereby subjecting the resin bound peptide tothe degradative effects of overlong acid exposure. With the vortexagitator there are no impeller type shear or abrasive effects on theresin beads. Those skilled in the art will understand that although inthe preferred embodiment the reaction vessel has been constructed in theshape of a right circular cylinder, other shapes can also be used,provided they are not antagonistic to the relatively smooth swirling ofthe fluid in the vessel, e.g. a shape such as that of a wine glassappears to have some desirable properties for the washing cycle. Also,for maximum efficiency, the apparatus is typically implemented withthree reaction vessels 15 while using only one concentrator vessel 13and one activator vessel 11, with the fluid distribution from theselatter two vessels appropriately valved to operate with each of thethree reaction vessels and their corresponding valve blocks 43 and 45.It should be understood, however, that each of these reaction vesselscorresponds to a separate sequential process for creating a peptide,i.e. the first peptide is formed in the first reaction vessel, then thesecond peptide is formed in the second reaction vessel, then the thirdpeptide is formed in the third vessel.

In order to monitor the progress of the synthesis in the reactionvessel, a resin sampler system 51 is provided. The sampler includes atube 53 connected into the side of the reaction vessel for extractingmaterials therefrom, the flow through the tube being controlled by acomputer controlled valve 55, typically a solenoid operated pinch valve.Tube 53 extends into the bottom portion of resin sampler reservoir 57which has a membrane 59, typically ZITEX™ located at the top. Alsoconnected to the top of reservoir 57, on the opposite side of membrane59, is a tube 61 which is connected to valve block 45. From the bottomof the reservoir extends another tube 65, which is controlled by a valve67 also typically a solenoid operated pinch valve (to achieve zero deadvolume), for collecting fractions from the reservoir.

The gas distribution system for achieving the desired transfers withinthe apparatus is illustrated in both FIGS. 1A and 1B, and consists ofthree gas manifolds, manifold C, manifold CC, and manifold D, andaccompanying regulators for controlling the distribution from bottlesand through the valve blocks. Also included are two smaller manifolds,manifold 70 for distributing DCM throughout the apparatus and manifold71 for distributing DMF to the activator vessel and the concentratorvessel. Gas flows throughout the system are controlled by the computersystem by means of solenoid operated valves such as valve 73; (e.g. suchas fluorocarbon valves are made by Angar, Incorporated.) and by thevalve blocks already discussed.

FIG. 2 shows a schematic representation of the computer system whichconsists of a microprocessor based microcomputer 81 having an arithmeticunit 87; a mass storage device 84, such as a floppy disk; a randomaccess memory (RAM) 83 for high speed; a hard copy output device 85,such as a printer; and a touch screen 82 which, in the preferredembodiment, operates as the only input device available directly to theuser. The system operates a switching apparatus 89, a switch being thebasic on/off device which the operator uses to control all the valves,the vortexers, the autodelivery system 90, and the heater 37. Typically,there is a one-to-one correspondence between devices and switches in thesystem so that each device corresponds to a particular switch number.For example, switches 0-63 may refer to valve numbers 0-63; the heatermay be controlled by switch 64, etc. Also to implement other elements ofautomatic control, the microcomputer 81 receives input signals from theliquid sensors in order to identify the times of gas-liquid andliquid-gas transitions, and it receives information from a bar codereader 108 located on the autodelivery system, for cross-checking theidentification of amino acids entering the synthesizer.

Shown in FIG. 3 is a top view of the autodelivery system. The system hasa guideway 101 which serves as a track to hold and control the directionof motion of an array of cartridges, such as cartridge 105. Each of thecartridges in the array contains an individual protected amino acid, andis placed in the array in the sequence that is desired in the peptide tobe synthesized. For convenience, the guideway is open for visualinspection of the array and is oriented to correspond to the peptidewith the carboxyl terminus on the right, which mimics the typical waypeptides are depicted in the literature and facilitates the comparisonof the sequence in the loaded guideway with that of the desired peptide.To hold the array of cartridges, a pressure block 103 is held againstthe last cartridge of the array by a spring reel 107 which is typicallyimplemented with a steel restorer tape 110 entrained over a pulley 109.This allows movement of the pressure block along the guideway whilestill providing a substantially constant force against the array ofcartridges, thus accommodating arrays of different lengths whichcorrespond to peptides of different lengths. Additionally, more than onepeptide can be synthesized from a single array of cartridges. On the endof the guideway opposite pressure block 103 is an ejector system 115driven by an air cylinder 113, for holding cartridges in a samplingposition 117 such as that shown for cartridge number 2, and for ejectingthe cartridges once the amino acids therein are educted. Position 111for cartridge number 1 illustrates the eject position of ejector system115, from which the spent cartridge falls down a shoot (not shown) andis disposed of. When the ejector returns to its normal position afterejecting, the constant force spring 107, acting through pressure block103, forces the next cartridge into delivery position.

Also included in the autodelivery system is a bar code reader 108. Inthe preferred embodiment, each cartridge is labeled with a bar codeunique to the kind of amino acid it contains. When a cartridgeprogresses down the guideway to the location of the bar code reader, thereader reads the bar code label and sends the information to thecomputer system. If the computer has been pre-set for a particularpolypeptide, it performs a consistency check to ensure that thecartridge is in the correct position in the sequence for thatpolypeptide. If the computer has not been pre-set for a particularpolypeptide, the system runs open loop and the computer uses theinformation from the bar code to call the synthesis protocol to be usedfor that particular amino acid in the cartridge and to record the aminoacid used. Also, each cartridge contains a stoichiometrically correctquantity of amino acid.

In order to educt amino acid from a cartridge, the system is providedwith two syringe needles (shown in FIG. 1), a needle 121 for supplyinggas pressure and for venting and a needle 123 for supplying DCM to thecartridge to dissolve the amino acid and for educting the dissolvedamino acid and DCM. The two needles are typically mounted vertically andconnected to an air cylinder (not shown) for moving the needles up andout of the way when a new cartridge is moved into place, and for drivingthe needles down through the top of cartridge for the mixing andeducting operations.

FIGS. 5a, 5b, and 5c show the details of the typical cartridge 105 usedin autodelivery system. In the preferred mode, the container isconstructed of blown, high density, polyethylene, and has a body portion130 of substantially rectangular cross-section capable of holding about7 ml. It also has a neck portion 133 onto which is attached aserum-finished cap 135 having an integral septum 137, which acts toprovide a positive seal of the cap to the neck. As illustrated in FIGS.4a and 4b, the dimension D1, (˜0.5 in.) is typically considerably lessthan dimension D₂ (˜1.120 in.) so that a relatively large number ofcartridges (up to 50) can be used in an array on guideway 101 withoutthe length of the array becoming unwieldy. Also, to promote properpositioning in the array, the cartridge has two substantially flatsurfaces 134 and 136 on each face.

The bottom of the cartridge is formed in the shape of two planes 140 and141 intersecting at an angle to form a vee-shaped trough. When thecartridge is in the sampling position in the autodelivery system, needle123 descends very close to the line of intersection 143 of the twoplanes, which corresponds to the locus of points having the lowestelevation in the cartridge, i.e. in the bottom of the cartridge. Theneedle being located near the lowest point in the cartridge helps toensure that all of the material in the cartridge is educted, therebymaking possible careful control of stoichiometry. In order to stabilizethe cartridge as it sits in guideway 101, a flange 145 extends acrossthe bottom of the cartridge in a direction perpendicular to the line ofintersection 143. The vee-bottomed trough and flange 145 make itpossible for the cartridge to stand unassisted in a stable uprightposition. The cartridge also includes an indentation 147 around itscircumference to promote precise placement of a bar code label to ensurethe accuracy of bar code reader 108 in reading the label.

FIG. 6 is a table listing the various dimensions of the bottle.

Illustrated in FIGS. 7a, and 7b are cutaway views of a typical liquidsensor used in the synthesizer. In the top view of FIG. 7b, the deviceis symmetric about the centerline CL, so the top half of FIG. 7bcorresponds to the bottom view of the top half of the device, and thebottom half of FIG. 7b corresponds to the top view of the bottom half ofthe device. The device is made up of a clothespin-like tube-holderhousing having a top portion 222 and a bottom portion 220, typicallyconstructed of glass-filled nylon or plastic, each of which has a groove229 with a double curvature extending across the width thereof toaccommodate a translucent tube. The double curvature is provided toenable the top and bottom portion to positively engage two differenttube diameters, which in the preferred embodiment are typicallyone-eighth or one-sixteenth of an inch in outside diameter andconstructed of TEFLON™. The top and bottom portions 222 and 220 aremounted by pegs 212, 213, 214, and 215, to two substrates, 209 and 211,respectively, which are typically constructed of printed circuit boardmaterial (phenolic). At the end opposite the tube-holder, the substratesare held a fixed distance apart by two rivets 240 and 241 ofsubstantially the same length. By providing a thickness of thetube-holder housing, from top and bottom, which is thicker than thelength of the rivets, the substrates provide a spring-like force to keepthe top and bottom portion of the housing together, thereby firmlyholding the tube in groove 229. Also to ensure accurate alignment of thetop and bottom portions, a key arrangement is provided with keys 225 and226, located on each side of the top portion which fit into holes 227(not shown because of the cutaway in FIG. 7b) and 228 located in bottomportion 220. In the side view of FIG. 7a, bottom portion 220 has beencut away to reveal a hole 250 in which is located a photodiode 270.Immediately opposite hole 250 across groove 229 is an identical hole 251located in top portion 222 for accommodating a photodetector 271, whichis used to detect the change in intensity of light received from thephotodiode when the interface between a liquid and gas, or between a gasand liquid moves down the tube held in groove 229, the change inintensity being due to the difference in focusing of the light rays dueto the difference in refractive properties of liquid and gas. Also, avoid 252 is provided in top portion 222 to accommodate a holder for thephotodetector, and a similar void 253 is provided in lower portion 220to accommodate a holder for the photodiode. Similarly a conduit 260through bottom portion 220 and conduit 261 through top portion 222provide paths for the electrical leads from the diode and detector,respectively, to solder pads 230 and 231 which are located at the endsof electrical runs 233 and 234, and to the outside generally for thedetector signal lead. Power is provided to the photodiode and thedetector via input terminals 235 and 236. Terminals 237 and 238 providea common ground for both the photodiode and the detector.

Synthesizer Operation

Synthesis of a peptide is initiated by first loading the reaction vesselwith resin, typically to which is attached the first amino acid in thesequence, and entering the desired amino acid sequence into thecomputer. The operator then loads the amino acid cartridges into theautodelivery system in the linear sequence or chain that corresponds tothe amino acid sequence of the desired desired peptide.

A cycle of activation begins when needles 121 and 123 puncture theseptum of the first cartridge, and needle 123 injects a calibratedamount of DCM. Gas sparges from needle 121 are used to mix and dissolvethe protected amino acid in the DCM. After dissolution the protectedamino acid is educted and transferred to the activator vessel. To assuretotal transfer of the protected amino acid, a second (and perhaps third)volume of DCM is added to the cartridge and then transferred to theactivator vessel. Then, based on the amino acid, 0.5 equivalent of DCCin DCM is delivered to the activator vessel and the solution is mixed byperiodic gas burps, e.g. argon or nitrogen. After a predetermined timeinterval sufficient for complete conversion of the amino acid to itssymmetric anhydride, the gas line of valve block 25 is opened and theDCM solution of the PSA is pressured out through valve block 23 to theconcentrator vessel. Frit 17 at the bottom of the activator vesselretains all of the DCU precipitate that is formed as a by-product in theactivation reaction. With software control, the PSA reaction times canbe individually adjusted for each amino acid to optimize PSA formationand for maximum precipitation of DCU. After transfer of the PSA/DCMsolution to the concentrator vessel, a volume of DMF is added. The venton the valve block 33 is then opened and an inert gas sparge throughvalve block 41 is commenced to volatilize the DCM. This is done withoutsignificantly reducing the original volume of DMF, which has asignificantly higher boiling point than DCM. Heat is supplied by bandheater 37 to replace heat lost by evaporation of the DCM. During thissolvent replacement process, different maximum internal temperatures canbe automatically adjusted to the unique thermal stabilities of thevarious protected amino acid PSA's by the use of thermistors.Concurrently with the solvent replacement process in the concentrator,the DCU precipitate in the activator is removed by successive washingswith an alcohol/DCM mixture via the top valve (valve block 23) andoverhead wash nozzle 19. The activator is finally washed withdichloromethane in preparation for the next PSA reaction. In theconcentrator vessel after the dichloromethane has been removed, thePSA/DMF solution is pressure transferred from the concentrator vessel tothe reaction vessel which contains the resin-boundalpha-amino-deprotected growing peptide chain.

The PSA/DMF that has been brought into the reaction vessel from theconcentrator vessel reacts with the deprotected alpha-amino function ofthe resin bound peptide for a period of time sufficient for reactioncompletion (typically greater than 99%), after which spent reagent andsolvent are washed out by successive solvent washes while using vortexagitation.

To begin a new cycle of synthesis in the reaction vessel it is firstnecessary to remove the alpha-amino protecting group of the last aminoacid which was attached to the chain. In the specific case of t-BOCprotected amino acids, a solution of trifluoroacetic acid (TFA) and DCM,typically 65% TFA, is pressure transferred to the reaction vessel andvortex agitation is periodically applied for effective mixing. After atime sufficient for total removal of the t-BOC-alphaamino protectinggroups (typically about 15 minutes), the fluid is pressured out throughvalve block 45 to waste. The resin is then washed rapidly with smallvolume increments of DCM introduced either through the top or bottomvalve while vortexing. Neutralization is effected by the introduction ofdiisopropylethylamine (DIEA) and DMF or DCM, vortexing, followed bypressure delivery to waste. Neutralization is usually repeated once. Theresin is then washed by successive additions of DCM (or DMF) in smallvolume increments through valve block 45 with the top valve (valve block43) open to waste, while vortexing (vortexing may be continuous orintermittent). After thorough washing of the amino-deprotected resin,the next amino acid PSA/DMF mixture is pressure transferred to thereaction vessel from the concentrator vessel.

To sample the resin during synthesis or on completion of the peptide,first valve 55 is opened and line 61 is opened through valve block 45which is vented to waste, while valve 67 is kept closed. The reactionvessel is then pressurized from the top while vortexing, forcing resinand the reaction solution into sample reservoir 57. This drives resinagainst the membrane 59. Valve 55 is then closed, valve 67 is opened,and the waste line of valve block 45 is closed. DCM is passed backthrough line 61 from valve block 45 clearing resin from the membrane anddepositing the resin/DCM mixture in a fraction collector 64. The samplereservoir and its accompanying tubing is then washed by closing valve67, venting the reaction vessel, and transferring DCM through line 61from valve block 45 through valve 55, and into the reaction vessel.

This integrated system allows for simultaneous operations in thereaction vessel and in the activator and concentrator vessels. Forexample, deprotection, neutralization, coupling, and washing operationscan occur in the reaction vessel at the same time that the next aminoacid PSA is being formed in the activator vessel. The concentratorvessel can be cleaned at the same time activation is occurring theactivator vessel, and the activator vessel can be cleaned while theconcentrator vessel is engaged in solvent replacement. This simultaneityof operations makes possible large economies in cycle time.

The system also allows the use of various synthesis methodologies.Although the approach described above has been for peptide synthesis byt-BOC-amino acid PSA's, alternative methods using protected amino acidPSA's, such as F-MOC, could also be readily implemented. Similarly,synthesis could be implemented by using other active carboxyl speciessuch as mixed anhydrides, active esters, acid chloride, and the like,utilizing the activator vessel and concentrator vessel to pre-form theactivated carboxyl species just prior to introduction to the reactionvessel, thus eliminating the need for storage reservoirs of activatedamino acid species which are maintained throughout the time frame of thepeptide synthesis.

As indicated earlier special coupling procedures are necessary forasparagine, glutamine, and arginine and can be initiated in theactivator and concentrator vessels. In these cases double couplings withhydroxybenzotriazole (HOBT) and DCC are generally required, whereequimolar HOBT and DCC in DMF or DMF/DCM are preequilibrated and thencombined with an equivalent of protected amino acid for reaction in thereaction vessel.

To achieve this result, one approach is to first transfer one equivalenteach of HOBT/DMF and DCC/DCM to the concentrator vessel through valveblocks 23 and 41. Then two equivalents of protected amino acid from anamino acid cartridge are transferred to the activator vessel inappropriate solvents (DMF/DCM). Following that, half of the material inthe activator vessel is transferred by time-pressure control to theconcentrator vessel (containing the pre-equilibrated HOBT/DCC/DMF/DCM)for activation, after which the activated mixture is transferred to thereaction vessel. Analogous activation is commenced for the secondcoupling near the end of the first coupling cycle by recharging theconcentrator vessel with a second equimolar mixture of HOBT/DCC,followed by addition of the second equivalent of amino acid from theactivator vessel.

Computer Software System

At the most basic level, software control of the apparatus is a matterof turning valves and other switched devices on and off at the propertimes to achieve the desired flows of the various materials from onecontainer or vessel to another. At the same time, many of the varioussteps in solid phase synthesis are quite repetitive and notextraordinary in number. Such a situation lends itself conveniently to amore sophisticated control concept aimed at functional control by theoperator rather than having the operator dictate in detail the workingsof individual valves to achieve a desired result. For example, mostoften the operator would rather command the system to transfer thecontents of the activator vessel to the concentrator vessel, rather thanformulate a more detailed series of commands such as: (1) check theconcentrator vessel to see if it is ready to receive; (2) open the venton valve block 33; (3) open valve blocks 23 and 41 at the connection ofthe transfer line between the vessels; (4) open gas valve to pressurizethe activator vessel; (5) shut off the valves after a signal from liquidsensor 39 indicates that the fluid has been transferred. To achieve thiskind of user-friendly approach and still maintain the capability oftotally independent control over each element of the apparatus, thesoftware control system is implemented through the touchscreen using aseries of menus, which serve to provide the operator with a wide gamutof possibilities, from one extreme of using the system in a completelyautomated mode to the other extreme of operating the system by switchingthe individual valves.

The control concept involved is that each individual coupling of anamino acid to the peptide chain corresponds to three complete cycles:one cycle in the activator vessel, one cycle in the concentrator vessel,and one cycle in the reaction vessel. Each of these cycles consists ofan ordered set of timed individual steps, each of which corresponds to afunction which can occur in that vessel. As a general definition, afunction can be considered as corresponding to a named set of switcheswhich are turned on simultaneously (the normal position of each switchbeing off). In practice it is advantageous to number the variousfunctions and to separate them by vessel. A function for example, mightbe DCM TO ACTIVATOR. Such a function requires a particular configurationof open valves in order for DCM to be delivered to the activator. In acycle of the activator, this function may appear several times, e.g.after the bulk of symmetric anhydride has been transferred to theconcentrator vessel it may be advantageous to wash the activator vesseland DCU precipitate several times with DCM to remove any residualsymmetric anhydride. Each time this function occurs it will correspondto a different step in the reaction cycle occurring in the activatorvessel, and similarly for other functions which are required in eachcycle. The net result is that each cycle in a vessel is a series ofsteps, with each step corresponding to a function associated with thatvessel. To better illustrate this concept, the individual control menuswill now be described.

FIG. 8 shows the main menu of the system as it appears on thetouchscreen, which corresponds to the bottom of a tree (shown in FIGS.17a and 17b) of various other more detailed menus. Each outlined blockcorresponds to an area on the touchscreen by which the series of menusin that tree is accessed. For example, touching the screen at the blocklabeled "PEPTIDE SEQ. EDITOR", initiates another display, FIG. 9,listing all of the amino acids, and allows the selection and display ofthe order of amino acids from N to C terminus appearing in the peptideto be synthesized by simply touching the block containing the name ofthe desired amino acid in the desired sequence.

Touching the screen at the block labeled "PEPTIDE CHEMISTRY EDITOR",when the main menu is displayed brings up the PEPTIDE CHEMISTRY EDITORmenu, FIG. 10, which allows the operator to create or edit either cyclespecifications in a particular vessel or the run file specification. Asan example, if it is elected to create or edit a new activator cycle, aCYCLE EDITOR menu corresponding to the activator vessel appears on thescreen. (See FIG. 11.) This menu enables the operator to change theorder of the functions involved in each cycle of the activator, andsimilarly for menus corresponding to the other vessels.

Generally, a given cycle has three time fields associated with it: arequired primary time, "TIME"; a time based on detector measurements,"MIN TIME" and "ERR MODE"; and an additional time added for particularsets of steps in the reaction vessel, "ADD TIME". TIME has severalpurposes. When liquid detection is not specified for the step, TIME isthe total time for the step. When liquid detection is specified, theprimary time is the "time out" or maximum time allowed before continuingregardless of whether or not the appropriate transition was detected bythe liquid sensor. Also, when liquid detection is specified, the usermust specify a minimum time, MIN TIME, before the appropriate transition(liquid to gas or gas to liquid) is to registered. It is important tonote another effect of TIME when detection is specified. When the sensorindicates the correct transition has occurred after the specifiedminimum time, the indicated function will be terminated, i.e., theswitches for that function will be turned off. However, the next stepwill not be initiated until the primary time has elapsed. This isnecessary to ensure proper alignment of the cycles in each vessel toobtain optimum throughput. ERR MODE is used in the event that detectionis specified and the specified transition is not seen before the primarytime has elapsed, e.g. if a valve is blocked or a particular reservoiris empty. This mode can be used to trigger an alarm or, in some cases,to effect a non-disastrous termination of the synthesis. ADD TIME refersspecifically to the reaction vessel only, and corresponds to the amountof time (in tenths of a second) to be added to each of the threeprevious fields as a function of amino acid number in the sequence ofthe peptide being synthesized. Since the occupied volume in the reactionvessel increases with each additional amino acid coupling, the steptimes in the reaction vessel also increase. For example, if the value 10is entered into this field, one second (10/10ths) will be added to theprimary time for the second amino acid to be coupled in the peptidesequence. For the third amino acid, the time would be increased by twoseconds, and so forth.

If it is desired to edit a RUN FILE, EDIT OLD in the PEPTIDE CHEMISTRYEDITOR is selected, displaying the RUN FILE EDITOR menu shown in FIG.12. The table displayed therein corresponds to what is called the staticrun file. This file designates three cycles (one each for the activatorvessel, the concentrator vessel, and the reaction vessel), for each oftwenty six amino acid possibilities, and allows independent adjustmentof the concentrator temperature for each amino acid. The twenty sixamino acid possibilities provided are comprised of the twenty standardamino acids, an additional four for specialized use as might be desiredby the operator, and a BEGIN cycle and an END cycle to allow independentcontrol of these points in the synthesis. This static run file is theresult of specifying the chemistry for each cycle through the variousCYCLE EDITORS which have already been described). In addition, allcycles designated by a particular run file must be resident on a disccurrently installed in the system, since the RUN FILE EDITOR will onlyallow choices from a list of resident cycles. Also, each of the threereaction vessels may synthesize from a different (or the same) run file,since, at the time a run is set up, the run file is specified for theparticular vessel in the REACTION VESSEL MONITOR (which will bediscussed later). Typically, the operator may create, edit, and store anumber of run files on a single disc (up to 20). It should also be notedthat although the system is designed to permit a different cycle foreach amino acid possibility, it has been found in practice that notnearly that many cycles are needed to provide efficient operations.

The next menu which will be discussed is the VESSEL FUNCTION EDITOR.This menu is accessed from the main menu, and operates at the most basiclevel of the synthesizer. It involves the individual instructionsrequired to accomplish a particular function in a particular vessel. Forexample, if it is desired to change the definition of a function or addor delete functions which are to occur in the activator vessel, theFUNCTION EDITOR shown in FIG. 13 corresponding to the activator vesselis called. Here, the entire set of functions for the activator can bereviewed and changed. As indicated earlier, a function is a named set ofswitches turned on simultaneously. These functions usually create achemical flow path through the system. For example, the function "DCC TOACTIVATOR" may be defined as switches 114 (DCC delivery valve), 119(pressurize DCC bottle), and 125 (open activator to waste. Also somefunctions describe mechanical or electrical actions only, such as"heater on", (one switch). The system is organized into three types offunctions: ON/OFF, TOGGLE ON, and TOGGLE OFF, specified as type 0, 1,and 2, respectively. An ON/OFF function turns switches on for a givenstep in a cycle only. At the end of that given step, all switches forthat function are cleared (turned off) before the next function isactivated. A TOGGLE ON function directs the machine to turn on certainswitches and leave them on until a corresponding TOGGLE OFF functionturns them off. Subsequent functions will not affect the state of theswitches turned on by the TOGGLE ON function. Hence, during creation andediting of functions, it is important that all TOGGLE ON functions havea corresponding TOGGLE OFF function. Examples of TOGGLE ON functions are"Heater on" for the concentrator vessel, or "VORTEX ON" for the reactionvessel.

Calling the REACTION VESSEL MONITOR from the main menu presents thescreen shown in FIG. 14, which lets the operator set up and monitor thesynthesis. Two response fields permit the operator to select between twomodes of operation, a first mode which automatically controls theapparatus based on the input to the PEPTIDE SEQ. EDITOR and a set ofpreprogrammed cycles called the dynamic run file; and a second modewhich operates based only on which cartridges (kinds of amino acids) areloaded into the autodelivery system and a set of preprogrammed cyclesfrom a designated static run file. If the first mode is chosen, thecomputer initiates a question as to whether the operator wishes tochange the dynamic run file. If so, the operator responds in theaffirmative and a screen as shown in FIG. 15 is displayed, correspondingto an editor for the dynamic run file. This file is generated initiallyinternally when the operator enters the desired peptide sequence intothe PEPTIDE SEQ. EDITOR. It is simply a table listing the sequence ofamino acids in the order to be synthesized, with the designated cyclesfor each amino acid as has already been programmed from the designatedstatic run file. This editor is designed so that the operator can alterthe cycles used for particular amino acids in the sequence depending onwhere they are located in the peptide chain, so that the operator hasposition dependent control over the chemistry. Unlike the static runfile, the dynamic run file is not stored on the disc.

Following mode selection in the REACTION VESSEL MONITOR, a series ofquestions is then generated to ensure that the apparatus is properly setup to begin operations, e.g. checks are made as to whether the reactionvessel is loaded, and whether the autoloader has the desired number andorder of amino acids. Following that series of inquiries, the finalinquiry is whether to begin or to stop operations. Also as part of thefunction of the REACTION VESSEL MONITOR, the status of the reactionvessel is displayed, including the name of the activated amino acid thatis currently undergoing reaction, the time that the synthesis of peptidewas initiated, and the time when the synthesis was completed. Inaddition, the current coupling is displayed and the sequence ofcouplings already completed can be displayed and reviewed by scrollingback and forth on the screen.

Calling the CYCLE MONITOR from the main menu brings up the screen shownin FIG. 16. Here the current status of each vessel is displayed in realtime in terms of several parameters. On the first line is listed foreach vessel the particular number of the amino acid in the sequence ofthe peptide which is currently active in that vessel, along with theabbreviation of the name of the particular amino acid. The second linelists the particular cycle name currently ongoing in each vessel. Thethird line lists which step in the sequence of steps of the particularcycle is currently being carried on in each vessel. The fourth linelists the number of the function and the function name corresponding tothe particular step listed for that vessel. Also the elapsed time intoeach cycle is listed as is the status of each of the liquid sensors.

Several other menus may also be selected from the main menu under thegeneral category of MACHINE/STATUS SELECTIONS. These include: RESERVOIRSTATUS which relates to the fact that the apparatus monitors the volumeused from each reservoir during each cycle, so that when a reservoirruns low an alarm is registered to inform the operator of the problem;INSTRUMENT CONFIG. which is used to display and set the configuration ofthe machine itself, e.g. time of day, power line--120 V at 60 Hz, etc.;SYSTEM SELF TEST which tests all of the electronic functions to theextent practicable; CONTROL AND TEST which allows manual control of theapparatus to the extent that the operator can manipulate each valve andswitch, one at a time, in order to facilitate debugging and to enablemanual intervention of synthesis if necessary; and DISK UTILITIES whichallows the operator to carry out customary disc functions necessary tothe operation of a computer system, e.g. setting up files, purgingfiles, and renaming files.

Another characteristic of the apparatus which is controlled by thesoftware is the simultaneity of operations in the reaction vessel, theconcentrator vessel, and the activator vessel. By noting the varioustimes required to carry out each step of a cycle in each vessel, anautomated optimization scheme, hereinafter called a cycle compiler, hasbeen developed to provide maximum efficiency in the synthesis of eachparticular peptide given a particular chemistry. To achieve thisefficiency it is important to recognize that for each vessel certainevents (functions) in time, in each cycle, determine when transfers cantake place and when the vessel is ready to begin the process for anothertransfer. For the activator vessel, these functions correspond to thebeginning of a transfer, BOTA; the end of a transfer, EOTA. particularpeptide, given a particular chemistry. To achieve this efficiency it isimportant to recognize that for each vessel certain events (functions)in time, in each cycle, determine when transfers can take place and whenthe vessel is ready to begin the process for another transfer. For theactivator vessel, these functions correspond to the beginning of atransfer, BOTA; the end of a transfer, EOTA.

Similarly for the concentrator cycle, the key functions includeidentifying when the vessel is ready to receive, RC; as well as thebeginning of a transfer, BOTC; the end of a transfer, EOTC. For thereaction vessel, these functions delineate when the vessel is ready toreceive, RR. Using these concepts, a graphic representation of a cycleas it takes place in each vessel can be depicted as illustrated in FIG.18. From this figure, it is apparent that one of the first criteria foroperation is that

    T.sub.BOTA =T.sub.RC and T.sub.BOTC =T.sub.RR ;

i.e. the concentrator vessel must be ready to receive when a transferfrom the activator vessel is begun, and the reaction vessel must beready to receive when a transfer from the concentrator vessel is begun.The next step then is to determine in which vessel operations shouldbegin first, in order to optimize the process. This can be accomplishedby calculating the left side delays (from FIG. 18 ) for each vessel:T_(DLA), T_(DLC), and T_(DLR). These left side delays pertain to thewaiting time allowed before preparing the particular vessel for thefunction that is to take place there, e.g. for the activator vessel,some waiting time may be allowed before transferring amino acid into thevessel and creating the symmetric anhydride, and for the reactionvessel, there may be some waiting time allowed before beginning thedeprotection of the resin-bound peptide chain. Once these left sidedelays are calculated, the shortest delay (i.e. the longest preparationtime) then corresponds to the cycle which should begin first, and themaster time should be equal to zero for that cycle. These various delayscan be calculated according to the following equations. First define##EQU1##

For example, setting the master time equal to zero at the beginning ofthe cycle shown in FIG. 18 might yield delay times such as T_(DLA) =0,T_(DLC) =5, and T_(DLR) =3, i.e. the activator vessel would begin firstat time T=0 the reaction vessel then would begin 3 seconds later, andthe concentrator vessel would begin at T=5 seconds. The next requirementfor optimal throughput involves calculating the minimum separation ofthese three cycles with the three cycles of the next coupling sequenceTo calculate this minimum, it is important to evaluate the right sidedelays for the first cycle: ##EQU2## and where T_(AT), T_(CA), andT_(RT) correspond to the total time for the activator cycle,concentrator cycle, and reaction vessel cycle, respectively.

These right side delays must then be combined with the various left sidedelays for the next cycle to arrive at a minimum separation for thethree cycles, i.e. ##EQU3## where T_(S).sbsb.c.spsb.2-cl is the minimumseparation between the first cycle and the second cycle, and T_(DLA2),T_(DLC2), and T_(DLR2) are the left side delays of the second cycle(calculated using the same technique as for the left side delays in thefirst cycle).

This minimum separation can then be translated into the waiting timesrequired to start the second cycle in each of the vessels, i.e. ##EQU4##where T_(WA2), T_(WC2), and T_(WR2) are the waiting times for theactivator vessel, the concentrator vessel, and the reaction vessel,respectively, from the last step of the first cycle to the first step ofthe second cycle. In order to achieve optimum efficiency one of T_(WA2),T_(WC2), and T_(WR2) must be zero, as was the case for left side delaysin the first cycle. FIG. 19 shows the results of the above calculationfor a series of three cycles

UTILITY OF THE INVENTION

The ability to deliver amino acid symmetric anhydrides of predeterminedintegrity to the reaction vessel enables most coupling reactions toproceed in yields exceeding 99% using just single couplings. Because ofthis very high yield at each step, it is possible to synthesize peptideswith very high overall efficiency fully automatically. The combinationof activator vessel and thermally jacketed concentrator vessel make itpossible to study and develop optimal conditions for maximal symmetricanhydride formation for individual protected amino acids, byindividually modifying stoichiometry, reaction times in DCM, temperatureduring DMF replacement of DCM, and the time frame during which thesolvent replacement process is executed. Thus, for each type of aminoacid it is possible to automatically implement conditions which willreproducibly deliver a maximal quantity of symmetric anhydride to thereaction vessel containing the peptide resin.

In the prior art with peptide synthesizers having no ability to formpreactivated amino acids, the user is obliged to stop at each reactioncycle after coupling to monitor the extent of coupling. Then with theobjective of improving the yield, second, or even third couplings can beeffected in cases where the first coupling reaction was relativelyinefficient. The result is automation of a single cycle of synthesis atthe preclusion of automation from cycle to cycle. Alternatively,existing synthesizers can be used automatically from cycle to cycle byutilzing multiple couplings with in situ activation in the reactionvessel containing the peptide resin. This results in relativelyinefficient couplings since the activation method cannot be uniquelyoptimized for individual amino acids in the presence of the reactingamino group of the growing peptide chain.

The results shown in FIGS. 20a and 20b illustrate the relative advantageof the claimed apparatus for the synthesis of the decapeptide AcylCarrier Protein (6574). The uppermost line is a graphical representationof the individual cycle yields for each amino acid addition during thepeptide chain assembly as performed on the apparatus of the invention.Except for glutamine (symbolized by Gln), all amino acid additions wereaccomplished by a fully automated sequence of single couplings:glutamine requires a special chemical protocol well known in the artthat consists of double coupling with HOBT (hydroxybenzotriazole)activation. All other couplings were done with Boc-amino acid symmetricanhydrides.

The lower line depicts results of the synthesis of the same peptide on aBeckman 990 Peptide Synthesizer using Boc-amino acid symmetricanhydrides pre-formed manually, off the instrument. Reference: RezaArshady, Eric Atherton, Derek Clive, and Robert C. Sheppard J Chem. Soc.Perkin Trans 1, (1981) 529-537. The results demonstrate that fullyautomated synthesis was precluded by the need to manually preform thesymmetric anhydrides, and that the manually preformed symmetricanhydrides were not optimally formed.

Some peptide amino acid sequences demonstrate unique, sequence specificcoupling problems where even second and third couplings, typically inDCM, fail to effect greater than 99% coupling yields, irrespective ofthe activated form of the amino acid (see W. S. Hancock, D. J. Prescott,P. R. Vagelos, and G. R. Marshall, J. Org. 38 (1973) 774). However,those skilled in the art recognize that sequence dependent incompletecouplings performed in poor solvents such as DCM would be much improvedif performed exclusively in more polar solvents such as DMF (see S.Meiseter, S. B. H. Kent, Peptides, Structure & Function, Proceedings ofthe 8th American Peptide Symposium, pps. (103-106). The Acyl CarrierProtein (65-74) decapeptide is such as known problem sequence and theexcellent results achieved on the claimed apparatus by coupling withBoc-amino acid symmetric anhydrides in DMF demonstrate one of the chiefadvantages of the apparatus: optimal formation of the activated aminoacid in DCM, but coupling of the activated amino acid in DMF. Thisautomatically executed method affords a general solution to thesynthesis of problem peptide sequences.

What is claimed is:
 1. A device for providing at least one precisevolume of fluid for use in a chemical process requiring deliver ofprecise volumes of reactant fluids, said device comprising:(a) twosubstantially parallel sides; (b) two other sides, symmetrically locatedopposite each other and connected to said two substantially parallelsides to form a box shape; (c) a bottom portion, connected to all fourof these sides and having an internal configuration of a vee-shapedtrough that has an apex aligned substantially perpendicular to saidparallel sides; (d) a top portion, having a septum therein and connectedto said sides to form a cartridge having a closed volume; and (e) aflange connected to said bottom portion, aligned substantially parallelto said two substantially parallel sides and located substantiallymidway between said substantially parallel sides, orthogonal to the apexof said trough; wherein (i) said flanges and (ii) a portion of anexterior surface of said bottom portion, adjacent to the apex of saidtrough, form a cross-shaped surface that, when placed in contact with atop surface of a horizontal, flat support surface, will support elements(a)-(e) in an orientation in which the top portion is vertically abovesaid bottom portion.
 2. A device as in claim 1 wherein all of elements(a)-(e) are part of a unitary body.